Interpretive Summary: The objective of this study was to develop a robust procedure to cryopreserve ram semen. The primary elements under investigation to develop this robust procedure were extender and freeze rate. Two commercially available semen extenders, Biladyl, and a Two Step Extender, were utilized because they have been previously used successfully in cattle, and also due to their straightforward preparation that lends them to easy field preparation. A range of prescribed freezing heights (4,5, and 6 cm) above the liquid nitrogen were investigated due to the difficulty seen in maintaining a constant level of liquid nitrogen in field conditions. Computer Assisted Sperm Analysis technology was utilized to measure and compare the pre-freezing and post-thaw motility characteristics. Biladyl produced significantly greater post-thaw total and progressive motility. The results show that the tested heights above the liquid nitrogen did not differ significantly for the post-thaw motility parameters for either extender. No significant differences between freezing rates at 4, 5, and 6 cm implies that technicians can utilize this protocol in field situations where liquid nitrogen levels are more likely to vary.

Technical Abstract:
Cryopreservation of germplasm is an important aspect in the conservation of genetic resources. However, for many species of animals the cryopreservation process has not been optimized. A challenge for conservation efforts is implementing cryopreservation methods that place no selection on the breed or individuals within breed for their ability to endure cryopreservation. To examine this challenge a 2x3x2 factorial experiment using Warhill sheep (a rare breed) was conducted. Experimental factors included: two tris-citrate-egg yolk extenders, Biladyl (Minitube of America) and Two-Step Extender (Continental Plastics); three freezing rates designated by height above LN2 (4, 5, and 6 cm); and two collection times for each ram. Six rams were collected via electro-ejaculation. Neat semen was processed using Certified Semen Services methods. Ejaculates were split and placed into either Biladyl or the Two-Step extender. Extenders were prepared by the manufacturers' instructions and clarified via centrifugation at 10,000 rpm for 10 minutes. Upon arrival to the lab the extended semen was evaluated using Computer Assisted Sperm Analysis (CASA). Semen was cooled to 5C, and frozen at the experimental heights. Samples were held for several weeks in LN2, and post-thaw analysis was performed after thawing at 37C. Data were analyzed using a mixed model: animal, collection and replication were considered random effects and extender and freeze height were fixed effects. Evaluation of variance components indicated that ram and collection nested within ram were significant and consistently the most important sources of variation in the model for CASA parameters. The pre-freeze CASA parameters indicated that the Two-Step Extender had significantly higher progressive motility(40.33 vs. 26.42%), progressive velocity (77.87 vs. 69.63 µm/s), beat cross frequency (31.61 vs. 26.83 Hz, ), straightness (84.92 vs. 78.83%), and linearity (59.58 vs. 50.96%), however Biladyl had a significantly higher lateral head displacement (5.88 vs. 5.01µm). Neither extender had a significant effect on pre-freezing total motility, path velocity, or track speed. Post-thaw analyses showed no significant freeze rate effects. Biladyl has significantly greater (p<.0001) post-thaw total and progressive motility (35.93 vs. 20.38%, and 19.69 vs. 11.09%, respectively). Freeze Rates were not a significant source of variation, which indicates that there is some latitude for temperature variation in cryopreserving rams with the tested extenders. Although rams and collection within rams were important sources of variation, Biladyl consistently provided better cryoprotection.